1. Evaluation of the anti-angiogenesis effect of MEK inhibition in thoracic cancers. As an extension of our previous work examining the effect of blocking EGFR-dependent signal transduction using the selective EGFR-RTKI PD153035 to mediate down-regulation of expression of pro-metastasis phenotypes in cultured thoracic cancer cells, we evaluated the effect of blocking MEK-mediated signaling on disrupting tumor angiogenesis. The MEK/ERK1/2 pathway is constitutively active in a panel of more than 15 cultured thoracic cancer cells even in the absence of serum. At low micromolar concentrations UO126 selectively abrogates MEK activity. Treating cultured thoracic cancer cells with UO126 results in inhibition of cell proliferation, clonogenicity as well as reduction of cell motility and invasion of Matrigel. UO126 (1.0 to 40 microM) significantly suppressed pro-angiogenesis cytokines VEGF, IL8 and PGE2 production by thoracic cancer cells. Moreover, EGF- and IL-1 induced robust upregulation of VEGF, IL8 and PGE2 is significantly blocked by UO126 (2.5 to 10 microM). This study demonstrates the antiangiogenesis property of MEK inhibitor via its ability to interfere with both cancer cell-derived pro-angiogenesis cytokines production as well as direct inhibition of endothelial cell function. Future study is being planned to evaluate the angiangiogenesis property of MEK inhibitor in an animal in vivo model. 2. Development of clinically applicable strategies to enhance the tumoricidal property of the recombinant apoptosis-inducing ligand Apo2L/TRAIL. My laboratory has focused a great deal of efforts to develop clinically applicable strategies to enhance the anticancer effect of the recombinant death-inducing protein ligand Apo2L/TRAIL (Tumor Necrosis factor-Related Apoptosis Inducing Ligand) for thoracic malignancies (primary cancers of the lung, the esophagus and the pleura). This project is part of an ongoing CRADA with Genentech, Inc. My laboratory has demonstrated that the majority of cultured thoracic cancer cells are intrinsically resistant to the cytotoxic effect of Apo2L/TRAIL. Exposure of these Apo2L/TRAIL-refractory cells to sublethal concentrations of cisplatin or paclitaxel (standard cytotoxic chemotherapeutic drugs), Trichostatin A (a histone deacetylase inhibitor) or Valproic acid (an anti-epileptic drug with histone deacetylase inhibitory activity) results in profound sensitization of these cells to Apo2L/TRAIL. Regardless of the class of agents used to sensitize cancer cells to Apo2L/TRAIL, the enhanced cytotoxicity by the drug combinations is profoundly inhibited by overexpression of Bcl2 and by the caspase 9 inhibitor z-LEHD-fmk, indicating the essential role of the mitochondria-mediated (type II pathway) death signaling cascade in mediating such effect. We further observed that the proteolytic activity of caspases 8,9 and 3 is high in combination treated cells. Over-expression of Bcl2 or blocking caspase 9 activity using z-LEHD-fmk not only abrogates caspase 9 and 3 activity but also the activity of the upstream caspase 8 suggesting the increased caspase 8 in combination-treated cells is most likely the result of the mitochondria-dependent positive feedback loop and not the result of increased DISC activity. This feedback loop-mediated activation of caspase 8 is essential for maintenance of activation of the caspase cascade and combination-induced cells death as blocking of caspase 6 (which is downstream of caspase 9 and upstream of caspase 8 and not related to caspase 3 activation) abrogates all caspase activation and treatment-induced cell death. First on the list of such compounds is a class of drug known as BH3 mimetic. These compounds, either naturally occurring or synthetic, share the common property of interacting with the BH3 binding pocket of the Bcl2/BclXL proteins and inhibit their anti-apoptotic function. We elected to evaluate the cytotoxic effect of the combinations of Apo2L/TRAIL and Gossypol, a phytochemical isolated from cottonseed oil and recently demonstrated to have BH3-mimetic property, or BH3I-2, a synthetic BH3-mimetic drug, in cultured thoracic cancer cells. Gossypol, as well as BH3I-2 synergistically interacts with Apo2L/TRAIL to induce massive cytotoxicity and apoptosis of cancer cells and not normal cells. Interstingly, we observed that certain cancer cells, while exquisitively sensitized to Apo2L/TRAIL by cytotoxic chemotherapeutics, are not sensitized by BH3 mimetics including BH3I-2 or Gossypol. Our current focus is to elucidate the molecular basis of BH3-mimetic-induced enhancement of Apo2L/TRAIL sensitivity in cultured thoracic cancer cells. 3. In addition to developing novel molecular therapeutic for thoracic cancer using Apo2L/TRAIL, our laboratory has also devoted significant efforts in studying cytotoxic gene therapy for thoracic cancers, particularly esophageal cancer using tumor-selective adenovirus vector as this kind of tumor is potentially amenable to gene therapy via direct intratumoral injection of therapeutic vectors using endoscopic approach. We have evaluated 3 generations of tumor-selective recombinant adenovirus expressing membrane-bound full length TRAIL protein. Tumor-selectivity of these viruses is achieved by employing the human telomerase reverse transcriptase (hTERT) promoter to control expression of the therapeutic TRAIL gene. Using flow cytometry and dual gating for GFP (TRAIL expressing cells) and Annexin V-PE (apoptotic cells), we also demonstrated the presence of strong bystander GFP-TRAIL-mediated cytotoxicity in cancer cells treated with the CDDP+AdVgTRAIL combination in vitro. Moreover, we further demonstrated that the profound cytotoxicity noted in combination-treated cells was mediated by recruitment of the type II mitochondria-dependent apoptosis signaling cascade as evidenced by abrogation of treatment-induced cytotoxicity by Bcl2 or by the selective caspase 9 inhibitor. Similar to chemotherapy+Apo2L/TRAIL treatment strategies described in section 3, the supra-additive activation of caspases 9,3 and more importantly caspase 8 following CDDP+AdVgTRAIL treatment was completely abrogated by overexpression of Bcl2 or by the selective caspase 9 inhibitor; indicating that the mitochondria-dependent death signaling pathway is essential in amplifying the caspase activation cascade. The preparation of a manuscript describing these findings is near completion and planned to be submitted to Cancer Gene Therapy journal. In the later part of FY 2006, we have performed comparative analysis of the tumoricidal efficacy of the tumor-selective oncolytic virus AdV-E1-TRAIL and the other two replication-defective virus AdVgTRAIL and ADV-RGD-TRAIL in vitro and in vivo. Due to its CMV-hTERT chimeric promotor system and its replication-competency, AdV-E1 vector mediates more robust transgene expression than the other two viral vector counterparts as determined by flow cytometric analysis for GFP fluorescence intensity using similar viral vectors expressing the GFP reporter gene instead of the therapeutic gene TRAIL. In SEG1 and SK4 EsC cells that are TRAIL-susceptible and permissive to adenovirus infection, AdV-E1-TRAIL mediated superior cytotoxicity to AdV-RGD-TRAIL or AdV-gTRAIL. In TRAIL-resistant EsC cells TE2 and TE12, AdV-E1-TRAIL offered a marginal advantage over the other two viruses on inducing cytotoxicity. In the presence of CDDP pretreatment used to enhance TRAIL cytotoxicity, AdV-E1-TRAIL was more potent than the others in mediating cytotoxicity, especially in TE12 cells